The overall goal of this project is to identify new compounds, as well as new pathways to discover new compounds for development of new treatments for the major AIDS- and cancer-related disseminated fungal infections. This is a competitive renewal of R01 AI27094 and expands our current work in the area of antifungal drug discovery, with an emphasis on the identification of mechanistically characterized and validated novel antifungal compounds. Our approach is to validate the unique lead compounds with antifungal and resistance-reversing properties that were identified in the previous project period, determine their mechanisms of action, and evaluate their in vivo efficacies against resistant strains. The specific aims of this project are: 1. Validate new lead compounds (already in hand) that show reversal of fluconazole resistance by using efflux pump assays and genomic approaches; 2. Determine mechanisms of action of lead antifungal compounds using genomic profiling approaches, and validate the molecular targets identified using genetic and biochemical strategies; 3. Evaluate the in vivo efficacy of selected lead compounds in murine models of systemic mycoses, including activity against resistant strains; and, 4. Identify other new lead compounds that exhibit unique mechanistic profiles. Successful completion of the proposed work will yield new prototype lead compounds that will serve as templates for development of new classes of antifungal agents, as templates for the development of new drugs to be used in combination with existing antifungal agents to reverse resistance to these agents, and as probes to identify novel mechanisms of action that can be exploited in the future search for other new derivatives. This information will expand the foundation of knowledge regarding the therapeutic control of these infections and the understanding of the basic biology of these pathogens.